SIMPL Is a Tumor Necrosis Factor-specific Regulator of Nuclear Factor-κB Activity

Abstract

Tumor necrosis factor ␣ (TNF␣) 1 is a pro-inflammatory cytokine that functions as an activator of the innate immune response. Produced mainly by activated macrophages, TNF␣ can influence cell survival or cell death (for review see Ref. 1). TNF␣-mediated activation of nuclear factor-B (NF-B)-dependent signaling is postulated to occur exclusively through the TNF receptor type I (TNF-RI). NF-B binds regulatory elements in the promoters of genes that encode mediators of the acute and chronic inflammatory response (for review see Refs. 2-4). Prototypical NF-B is a heterodimer composed of a 50-kDa subunit (NF-B/p50) and a 65-kDa subunit (RelA/p65). NF-B activity is most likely regulated at two levels, cytoplasmic sequestration and direct removal from DNA. An additional protein family, inhibitors of B (IB), is involved in both regulatory processes (for review see Refs. 5 and 6).Inactivation of IB protein activity is the result of rapid stimulus-dependent phosphorylation on two critical amino-terminal serine residues (7-10). The phosphorylations are a prerequisite for ubiquitin-targeted proteasome-dependent degradation of IB. A large multiprotein complex that includes at least the IB kinases (IKK␣ and IKK␤) and the scaffolding protein IKK␥ (NEMO) has been described previously (11-18). IKK␣ and IKK␤ are responsible for IB phosphorylation and are themselves substrates of phosphorylation events necessary for IKK activation (for review see Refs. 19 and 20).The IL-1 receptor-associated kinase (IRAK) was identified as a protein associated with the type I IL-1 receptor (21), whereas the mouse homologue of IRAK, mouse pelle-like kinase (mPLK), was identified independently (22) in a cDNA library screened for kinases related to the Drosophila pelle kinase (23). We have demonstrated that IRAK/mPLK protein kinase activity is critical for TNF-RI signaling and placed mPLK/IRAK in a novel signal transduction pathway through which TNF-RI activates NF-B-dependent gene expression (24). Although the IRAK/mPLK protein may be required for IL-1-induced NF-B activity, IRAK/mPLK catalytic activity is not required for IL-1-dependent signaling (24 -26). In mPLK/IRAK null fibroblasts IL-1 as well as in TNF-induced NF-B, DNA binding activity is significantly attenuated (27). In this report we describe a novel signaling molecule that associates with the mouse pelle-like kinase (SIMPL) and demonstrate that SIMPL is required for TNF-RI-dependent activation of NF-B activity. EXPERIMENTAL PROCEDURESLibrary Screening-DNA fragments encoding different regions of the IRAK/mPLK protein (21, 24) were subcloned into pGBDU and were transformed into the yeast strain PJ69-4A (28). Transformants were selected on SD/ϪUra, SD/ϪAde, SD/ϪHis to test for autoactivation of the reporter genes. A pGBDU construct encoding amino acids 1-533 of mPLK (pGBDU-mPLK⌬534 -710) that did not activate transcription in the absence of a bait plasmid was subjected to a two-hybrid analysis. A day 17 mouse embryo Matchmaker cDNA library (CLONTECH, Palo Alto, CA) was transformed into PJ69...

Cutting Edge: Mouse Pellino-2 Modulates IL-1 and Lipopolysaccharide Signaling

“…2A) which suggests the mPLK/IRAK-1 and Pln-2 proteins interact in mammalian cells. Another mPLK/IRAK-1-interacting protein is SIMPL, which is required for TNF-␣-dependent activation of NF-B-dependent gene expression (19). A search of the Drosophila database (FlyBase) failed to identify a Drosophila SIMPL homolog, so it was of interest to determine whether pellino-2 and SIMPL interact and form complexes.…”

“…Ectopic expression of pellino-2 in mouse embryonic fibroblasts did not result in activation of a heterologous reporter composed of the firefly LUC cDNA under the control of the NF-B-dependent IL-8 gene promoter (data not shown). In contrast to pellino-2, ectopic expression of either mPLK/IRAK-1 or SIMPL leads to a modest increase in IL-8 promoter activity (13,19). An analysis of a mPLK/IRAK-1 nulligenic demonstrated a requirement for mPLK/IRAK-1 in TNF-␣ and IL-1-dependent NF-B DNA binding activity (20).…”

“…The human embryonic kidney (HEK) cells (293-EBNA) and the C3H10T1/2 mouse embryo fibroblast cell line were maintained as described previously (19). Transfections were performed with the indicated DNAs using FuGENE-6 (Roche, Basel, Switzerland) according to the manufacturer's recommendations.…”

IRAK1BP1 inhibits inflammation by promoting nuclear translocation of NF-κB p50

“…These data suggest that the effect of IRAK1BP1 occurs through a general inhibitory role in inflammatory gene transcription rather than acting at the level of receptor-proximal signaling events. Although we previously demonstrated an inhibitory role for IRAK1BP1 in the transcription of several proinflammatory cytokines from their endogenous loci following TLR, IL-1R, and TNF-R stimulation (17), another report suggested that overexpression of IRAK1BP1 increased activation of NF-κB reporter plasmids following TNF-α stimulation (18). To broaden the list of target genes for IRAK1BP1, we extended our analysis to include the antiinflammatory cytokine IL-10.…”

“…Finally, its role in LPS tolerance assures that continued stimulation does not lead to unchecked cytokine release. In addition to its transcriptional profile in activated macrophages, previous work also has shown that IRAK1BP1 is constitutively expressed at high levels in the mouse brain and testis, two immune-privileged organs, suggesting that its expression also may be used to modulate the effect of inflammatory mediators such as IL-1 and TNF in specific tissues where their potentially harmful effects may outweigh their immune-activating benefits (18).…”